Elena Gabanelli

Epidemiological history and phylogeography of West Nile virus lineage 2

West Nile virus (WNV) was first isolated in Uganda. In Europe WNV was sporadically detected until 1996, since then the virus has been regularly isolated from birds and mosquitoes and caused several outbreaks in horses and humans. Phylogenetic analysis showed two main different WNV lineages. The lineage 1 is widespread and segregates into different subclades (1a-c). WNV-1a includes numerous strains from Africa, America, and Eurasia. The spatio-temporal history of WNV-1a in Europe was recently described, identifying two main routes of dispersion, one in Eastern and the second in Western Europe. The West Nile lineage 2 (WNV-2) is mainly present in sub-Saharan Africa but has been recently emerged in Eastern and Western European countries. In this study we reconstruct the phylogeny of WNV-2 on a spatio-temporal scale in order to estimate the time of origin and patterns of geographical dispersal of the different isolates, particularly in Europe. Phylogeography findings obtained from E and NS5 gene analyses suggest that there were at least two separate introductions of WNV-2 from the African continent dated back approximately to the year 1999 (Central Europe) and 2000 (Russia), respectively. The epidemiological implications and clinical consequences of lineage 1 and 2 cocirculation deserve further investigations.

Enigmatic origin of hepatitis B virus: An ancient travelling companion or a recent encounter?

Hepatitis B virus (HBV) is the leading cause of liver disease and infects an estimated 240 million people worldwide. It is characterised by a high degree of genetic heterogeneity because of the use of a reverse transcriptase during viral replication. The ten genotypes (A-J) that have been described so far further segregate into a number of subgenotypes which have distinct ethno-geographic distribution. Genotypes A and D are ubiquitous and the most prevalent genotypes in Europe (mainly represented by subgenotypes D1-3 and A2); genotypes B and C are restricted to eastern Asia and Oceania; genotype E to central and western Africa; and genotypes H and F (classified into 4 subgenotypes) to Latin America and Alaska. This review summarises the data obtained by studying the global phylodynamics and phylogeography of HBV genotypes, particularly those concerning the origin and dispersion histories of genotypes A, D, E and F and their subgenotypes. The lack of any consensus concerning the HBV substitution rate and the conflicting data obtained using different calibration approaches make the time of origin and divergence of the various genotypes and subgenotypes largely uncertain. It is hypothesised that HBV evolutionary rates are time dependent, and that the changes depend on the main transmission routes of the genotypes and the dynamics of the infected populations.

Spatial and Temporal Dynamics of Hepatitis B Virus D Genotype in Europe and the Mediterranean Basin

Hepatitis B virus genotype D can be found in many parts of the world and is the most prevalent strain in south-eastern Europe, the Mediterranean Basin, the Middle East, and the Indian sub-continent. The epidemiological history of the D genotype and its subgenotypes is still obscure because of the scarcity of appropriate studies. We retrieved from public databases a total of 312 gene P sequences of HBV genotype D isolated in various countries throughout the world, and reconstructed the spatio-temporal evolutionary dynamics of the HBV-D epidemic using a Bayesian framework. The phylogeographical analysis showed that India had the highest posterior probability of being the location of the tree root, whereas central Asia was the most probable location of the common ancestor of subgenotypes D1–D3. HBV-D5 (identified in native Indian populations) diverged from the tree root earlier than D1–D3. The time of the most recent common ancestor (tMRCA) of the tree root was 128 years ago, which suggests that the common ancestor of the currently circulating subgenotypes existed in the second half of the XIX century. The mean tMRCA of subgenotypes D1–D3 was between the 1940s and the 1950–60s. On the basis of our phylogeographic reconstruction, it seems that HBV-D reached the Mediterranean area in the middle of the XX century by means of at least two routes: the first pathway (mainly due to the spread of subgenotype D1) crossing the Middle East and reaching north Africa and the eastern Mediterranean, and the second pathway (closely associated with D2) that crossed the former Soviet Union and reached eastern Europe and the Mediterranean through Albania. We hypothesise that the main route of dispersion of genotype D was the unsafe use of injections and drug addiction.

Bayesian Phylogeography of Crimean-Congo Hemorrhagic Fever Virus in Europe

Crimean-Congo hemorrhagic fever (CCHF) is a zoonosis mainly transmitted by ticks that causes severe hemorrhagic fever and has a mortality rate of 5-60%. The first outbreak of CCHF occurred in the Crimean peninsula in 1944-45 and it has recently emerged in the Balkans and eastern Mediterranean. In order to reconstruct the origin and pathway of the worldwide dispersion of the virus at global and regional (eastern European) level, we investigated the phylogeography of the infection by analysing 121 publicly available CCHFV S gene sequences including two recently characterised Albanian isolates. The spatial and temporal phylogeny was reconstructed using a Bayesian Markov chain Monte Carlo approach, which estimated a mean evolutionary rate of 2.96 x 10-4 (95%HPD=1.6 and 4.7 x 10-4) substitutions/site/year for the analysed fragment. All of the isolates segregated into seven highly significant clades that correspond to the known geographical clades: in particular the two new isolates from northern Albania clustered significantly within the Europe 1 clade. Our phylogeographical reconstruction suggests that the global CCHFV clades originated about one thousand years ago from a common ancestor probably located in Africa. The virus then spread to Asia in the XV century and entered Europe on at least two occasions: the first in the early 1800s, when a still circulating but less or non-pathogenic virus emerged in Greece and Turkey, and the second in the early 1900s, when a pathogenic CCHFV strain began to spread in eastern Europe. The most probable location for the origin of this European clade 1 was Russia, but Turkey played a central role in spreading the virus throughout Europe. Given the close proximity of the infected areas, our data suggest that the movement of wild and domestic ungulates from endemic areas was probably the main cause of the dissemination of the virus in eastern Europe.

Reconstruction of the evolutionary dynamics of hepatitis C virus subtypes in Montenegro and the Balkan region

More than 20 million hepatitis C virus (HCV) carriers live in the countries of the Eastern Mediterranean. We determined HCV genotype distribution among chronically infected patients in Montenegro and investigated the phylodynamics and phylogeography of the most represented HCV subtypes. The HCV-NS5b sequences of the Montenegrin patients were compared with sequences isolated in different known localities of the Mediterranean area, Europe and Asia. A Bayesian approach was used in order to allow the simultaneous estimate of the evolutionary rate, time-scaled phylogeny, demography and ancestral spatial status. The most frequent HCV subtypes among the Montenegrin patients, were 1b (34.7%) and 3a (24.7%), but there was also a significant prevalence of 1a and 4d (19.5%). Subtype 3a was significantly more frequent among younger patients and intravenous drug users (IDUs), whereas subtype 1b was more frequently associated with iatrogenic exposure and older ages. The spatio-temporal analysis of the epidemic suggested that HCV-1b penetrated Europe at the beginning of the XX century, probably through Greece and Cyprus and in the 1920s reached Montenegro, where there was an exponential increase in the effective number of infections between the 1950s and 1970s. The phylogeographic and phylodynamic analysis of HCV 3a showed that its most probable origin was in the Indian sub-continent (Pakistan in our reconstruction) about 300years ago. The evolutionary dynamics analysis showed that HCV-3a reached Montenegro more recently in the late 1970s and underwent multi-phasic growth still persisting. Our data suggest multiple introduction of HCV subtypes in the area, supported by different causes of dispersion: adverse social conditions and unsafe medical practices for HCV-1b and i.v. drug use for HCV-3a.

Evolutionary dynamics of HBV‐D1 genotype epidemic in Turkey

Hepatitis B virus (HBV), is the leading cause of liver diseases infecting an estimated 240 million persons worldwide. The HBV prevalence rates are variables between different countries, with an high level of endemicity in the south‐eastern part of Europe. Seven main HBV‐D subgenotypes have been described until now (D1–D7). Turkey, seems to have played an important role in the penetration of HBV‐D1 in the Mediterranean area. The importance of Turkey in the European epidemiology of HBV is also suggested by the observation that the highest spread of HBV infection in the Continent are reported in Turkey with Romania, Bulgaria, Greece, Albania and some southern regions of Italy. In this paper the molecular epidemiology and the epidemiological history of HBV‐D in Turkey was studied, by characterizing 34 new Turkish isolates and performing a phylogeographic reconstruction. By using a phylodynamic and phylogeographic Bayesian approach, the analysis suggested that HBV‐D1 originated in Turkey about in the early 1940s. The large prevalence of D1 in comparison to the other subgenotypes in Turkey confirms the importance of this Country as epidemiological reservoir of HBV‐D1 dispersion. The phylogeny suggests that after each initial introduction of the virus in a specific population, separate transmission clusters have been evolving along independent phylogenetic lineages. Better characterization and continuous monitoring of such groups are going to be crucial to understand in detail the epidemiology of HBV‐D1 subgenotype in Turkey and to assess the efficacy of prevention, vaccination and therapy in controlling the epidemic. J. Med. Virol. 86:109–116, 2014.

Molecular analysis of hepatitis B virus in Bulgaria

Hepatitis B virus infection is a global health problem. Based on the sequence divergence of the entire genome, hepatitis B virus has been classified into eight genotypes which have a characteristic geographic distribution. To date, no data are available on the molecular epidemiology of hepatitis B virus in Bulgaria. The aim of the present study was to reconstruct the epidemiological history of HBV genotypes/subgenotypes circulating in Bulgaria using a phylodynamic approach and a Bayesian statistical inference framework. Sequence analysis of the HBsAg/Reverse Transcriptase overlapping genomic regions revealed that D1 and A2 were the subgenotypes detected most frequently in the patients examined. The tMRCA estimations of the few HBV D1 Bulgarian significant clades dated back to 23–27 years ago, corresponding to the early 1980s. The HBV A2 Bulgarian sequences fell into two closely related supported clusters dated to 2003 and 1996 years, respectively, suggesting a more recent introduction of subgenotype A2 into Bulgaria. The study provides new information about the HBV subgenotypes in Bulgaria. J. Med. Virol. 85:49–54, 2012.

Reconstruction of the Evolutionary Dynamics of the A(H1N1)pdm09 Influenza Virus in Italy during the Pandemic and Post-Pandemic Phases

The aim of this study was to reconstruct the evolutionary dynamics of the A(H1N1)pdm09 influenza virus in Italy during two epidemic seasons (2009/2010 and 2010/2011) in the light of the forces driving the evolution of the virus. Nearly six thousands respiratory specimens were collected from patients with influenza-like illness within the framework of the Italian Influenza Surveillance Network, and the A(H1N1)pdm09 hemagglutinin (HA) gene was amplified and directly sequenced from 227 of these. Phylodynamic and phylogeographical analyses were made using a Bayesian Markov Chain Monte Carlo method, and codon-specific positive selection acting on the HA coding sequence was evaluated. The global and local phylogenetic analyses showed that all of the Italian sequences sampled in the post-pandemic (2010/2011) season grouped into at least four highly significant Italian clades, whereas those of the pandemic season (2009/2010) were interspersed with isolates from other countries at the tree root. The time of the most recent common ancestor of the strains circulating in the pandemic season in Italy was estimated to be between the spring and summer of 2009, whereas the Italian clades of the post-pandemic season originated in the spring of 2010 and showed radiation in the summer/autumn of the same year; this was confirmed by a Bayesian skyline plot showing the biphasic growth of the effective number of infections. The local phylogeography analysis showed that the first season of infection originated in Northern Italian localities with high density populations, whereas the second involved less densely populated localities, in line with a gravity-like model of geographical dispersion. Two HA sites, codons 97 and 222, were under positive selection. In conclusion, the A(H1N1)pdm09 virus was introduced into Italy in the spring of 2009 by means of multiple importations. This was followed by repeated founder effects in the post-pandemic period that originated specific Italian clades.

Different evolutionary trends of swine H1N2 influenza viruses in Italy compared to European viruses

European H1N2 swine influenza viruses (EU H1N2SIVs) arose from multiple reassortment events among human H1N1, human H3N2, and avian influenza viruses. We investigated the evolutionary dynamics of 53 Italian H1N2 strains by comparing them with EU H1N2 SIVs. Hemagglutinin (HA) phylogeny revealed Italian strains fell into four groups: Group A and B (41 strains) had a human H1 similar to EU H1N2SIVs, which probably originated in 1986. However Group B (38 strains) formed a subgroup that had a two-amino acid deletion at positions 146/147 in HA. Group C (11 strains) contained an avian H1 that probably originated in 1996, and Group D (1 strain) had an H1 characteristic of the 2009 pandemic strain. Neuraminidase (NA) phylogeny suggested a series of genomic reassortments had occurred. Group A had an N2 that originated from human H3N2 in the late 1970s. Group B had different human N2 that most likely arose from a reassortment with the more recent human H3N2 virus, which probably occurred in 2000. Group C had an avian-like H1 combined with an N2 gene from one of EU H1N2SIVs, EU H3N2SIVs or Human H3N2. Group D was part of the EU H3N2SIVs clade. Although selection pressure for HA and NA was low, several positively selected sites were identified in both proteins, some of which were antigenic, suggesting selection influenced the evolution of SIV. The data highlight different evolutionary trends between European viruses and currently circulating Italian B strains and show the establishment of reassortant strains involving human viruses in Italian pigs.

Recurrence of another hepatitis B virus escape mutant comes back in a patient infected with HIV and low CD4+ count

The case of an HIV‐positive patient who cleared formerly acute HBV infection and experienced HBV exacerbation before starting antiretroviral therapy (ART) followed by a severe elevation of liver enzymes one month after the introduction of ART is reported. Laboratory tests revealed the presence of a subgenotype A2 carrying at least seven different mutations within the S protein and high lopinavir plasma concentrations. Irrespectively of the many possible mechanisms of HBV reactivation in patients infected with HIV (escape mutants selection, host immune deficiency progression, antiretroviral‐related immune reconstitution, glucocorticosteroids, chemotherapy), the message is that HIV patients should be monitored strictly not only by serological and virological assays to recognize early HBV reactivation, but also for drug over‐exposure. J. Med. Virol. 86:97–101, 2014.